Process for microencapsulating cells

ABSTRACT

Oxalate-degrading enzymes and bacteria were encapsulated for both enteric and intraperitoneal administration. We have shown that via alginate microencapsulation of Oxalobacter formigenes, enzymatic activity was retained for several months. A new process was developed which strengthened the aliginate microcapsules and their tolerance to citrate treatment. Much smaller (30-50 μm) aliginate microcapsules were made for injection as means of impantation. For oral administration, multi-encapsulated microspheres of cellulose acetate phthalate in poly-2-vinylpyridine (pKa=3.5) were prepared to protect the enzymes from gastric juices.

This invention was made with Government support under Grant No.NIH5PO1DK20586-14awarded by the National Institutes of Health. TheGovernment has certain rights in this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of application Ser. No. 08/192,212, filedFeb. 3, 1994 now U.S. Pat. No. 5,648,099; which is a division ofapplication Ser. No. 07/880,866, filed May 11, 1992, now U.S. Pat. No.5,286,495.

BACKGROUND OF THE INVENTION

Microencapsulation is the process of enveloping certain drugs, enzymes,toxins, or other substances in polymeric matrices. It can be used incontrolled release or delayed release of drugs. The many applications,available matrices, and techniques are extensively covered elsewhere(see, for example, Chang, T. M. S. [1977] Biomedical applications ofimmobilized enzymes and proteins, Vols. 1-2, New York: Plenum Press;Deasy, P. B. (ed.) [1984] "Microencapsulation and related drugprocesses," In J. Swarbrick (ed.), Drugs and the pharmaceuticalsciences: Vol. 20. Microencapsulation and related drug processes, NewYork: Marcel Dekker, Inc.; McGinity, J. W. [1989] "Aqueous polymericcoatings for pharmaceutical dosage forms," Drugs and the PharmaceuticalSciences 36; Nixon, J. R. (ed.) [1976] "Microencapsulation," In J.Swarbrick (ed.) Drugs and the pharmaceutical sciences: Vol. 3, New York,Marcel Dekker, Inc.).

Polymeric matrix microencapsulation of microorganisms is a relativelynew technology which has potentially major implications in the treatmentof various afflictions. Examples of afflictions in which treatmentinvolving microcapsules could be advantageous are diabetes and urinarystone diseases. Insulin dependent diabetes mellitus (IDDM) is a severedisease which afflicts millions of Americans, causing substantialdisruption of lifestyle and often resulting in severe health problems.The exact causes of IDDM have remained largely a mystery, despite yearsof intensive research on this disease. It is now widely recognized thatIDDM is an autoimmune condition whereby the body's natural immunologicaldefenses destroy the β-cells of the pancreas. β-cells are responsiblefor the production of insulin, and, once a substantial portion of theβ-cells are destroyed, those individuals afflicted with the disease mustrely on exogenous sources of insulin, usually in the form of injections.The success of pancreas or islet cell transplantations is very limitedbecause of immune responses typically mounted by the recipient againstthe foreign cells.

Urolithiasis, or urinary stone disease, is a common urinary tractproblem afflicting more than 10% of the U.S. population. Urinary tractstones are usually classified according to their composition, with themost frequently encountered (70%) being the calcium stone composed ofcalcium oxalate alone or calcium oxalate mixed with calcium phosphate.Although precipitation of calcium oxalate depends on a urine saturatedwith both calcium and oxalate ions in a metastable state, it has beenargued that the oxalate ion concentration is more significant in theformation of urinary calcium oxalate stones. Thus, the management ofoxalate in individuals susceptible to urolithiasis would seem especiallyimportant. The majority of oxalate in plasma and urine is derived fromthe endogenous metabolism of ascorbic acid, glyoxylate, and to a lesserdegree, tryptophan. In addition, between 10% and 20% of the urinaryoxalate is absorbed from the diet, especially through ingestion of leafyvegetables and plant materials, although there is disagreement in theliterature about the relative amounts of diet and endogenous oxalate.Ingestion of ethylene glycol, diethylene glycol, xylitol, and excessascorbic acid can lead through metabolic conversions to disorders ofexcess oxalate. Use of methoxyfluraneas an anaesthetic can also lead tooxalosis. Aspergillosis, infection with an oxalate-producing fungus, canlead to production and deposition of calcium oxalate. Other causes ofexcess oxalic acid include renal failure and intestinal disease.

It is believed that lowering the oxalate levels in the plasma, andsubsequently the urine, would decrease the incidence of calcium oxalatestone formation. Unfortunately, there are no known naturally occurringoxalate degrading or metabolizing enzymes in vertebrates. Catabolism ofoxalic acid appears restricted to the plant kingdom.

Hyperoxaluria can also be related to genetic disorders. Primaryhyperoxaluria is a general term for an inherited disorder which revealsitself in childhood and progresses to renal failure and frequently deathin adolescence. It is characterized by high urinary excretion of oxalateand recurring calcium oxalate kidney stones. Primary hyperoxaluriasconsist of two rare disorders of glyoxylate and hydroxypuruvatemetabolism. There are no satisfactory treatments for the two types ofprimary hyperoxaluria. Hemodialysis and renal transplantation have notbeen successful in halting the progress of this disease. Controlled diethas also failed to stop the complications of primary hyperoxaluria.Primary hyperoxaluria eventually leads to other abnormalities such asurolithiasis, nephrocalcinosis with renal failure, systemic oxalosis,and oxalemia.

Oxalate toxicity can also cause livestock poisoning, due to grazing onoxalate-rich pastures. Ingestion of oxalate-richplants such as Halogetonglomeratus, Bassia hyssopifolia, Oxalis pes-caprae, and Setariasphacelata, or grains infected with the oxalate-producing fungiAspergillus niger, has been reported to cause oxalate poisoning in sheepand cattle. Chronic poisoning is often accompanied by appetite loss andrenal impairment. Acute toxicity can lead to tetany, coma, and death(Hodgkinson, A. [1977] Oxalic acid in biology and medicine, London:Academic Press, pp. 220-222).

Three mechanisms for oxalate catabolism are known: oxidation,decarboxylation, and activation followed by decarboxylation (Hodgkinson,A. [1977], supra at 119-124). Oxalate oxidases are enzymes that arefound in mosses, higher plants, and possibly fingi which catalyze theoxidation of oxalate to hydrogen peroxide plus carbon dioxide: (COOH)₂+O₂ →2CO₂ +H₂ O₂. Oxalate decarboxylases are enzymes which produce CO₂and formate as products of oxalate degradation. An O₂ -dependent oxaiatedecarboxylase found in fungi catalyzes the decarboxylation of oxalicacid to yield stoichiometric quantities of formic acid and CO₂ : (COOH)₂→CO₂ +HCOOH. Varieties of both aerobic and anaerobicbacteria can alsodegrade oxalic acid. An activation and decarboxylation mechanism is usedfor degradation of oxalate in Pseudomonas oxalaticus and other bacteria.The many pathways leading to oxalate are discussed elsewhere(Hodgkinson, A. [1977] supra; Jacobsen, D. et al. [1988] AmericanJournal of Medicine 84:145-152).

Oxalobacter formigenes is a recently described oxalate-degradinganaerobic bacterium which inhabits the rumen of animals as well as thecolon of man (Allison, M. J. [1985] Arch. Microbiol. 141:1-7). O.formigenes OxB is a strain that grows in media containing oxalate as thesole metabolic substrate. Other substrates do not appear to support itsgrowth. The degradation of oxalate catalyzed by the bacterial enzymeresults in CO₂ and formic acid production (Allison [1985], supra).

Recently, research has focused on matrices used to encapsulate cells andorganisms. The use of alginate gel technology to formulate agriculturalproducts, pesticides, and food items has been disclosed. For example,U.S. Pat. No. 4,053,627 describes the use of alginate gel discs formosquito control; U.S. Pat. No. 3,649,239 discloses fertilizercompositions; and U.S. Pat. No. 2,441,729 teaches the use of alginategels as insecticidal as well as candy jellies. In addition, U.S. Pat.Nos. 4,401,456 and 4,400,391 disclose processes for preparing alginategel beads containing bioactive materials, and U.S. Pat. No. 4,767,441teaches the use of living fungi as an active material incorporated in analginate matrix.

The most usual hydroxyl polymers used for encapsulating biomaterials arealginate, polyacrylamide, carrageenan, agar, or agarose. Of these,alginate and carrageenan are the only ones which can be manufacturedsimply in spherical form with encapsulated material. This is done byionotropic gelling, i.e., the alginate is dropped down into a calciumsolution and the carrageenan into a potassium solution. However, theresulting beads are stable only in the presence of ions (calcium andpotassium, respectively).

The use of ultrasonic nozzles has offered a new way of making smallermicrospheres with very good control over the size of the droplets(Ghebre-Sellassie, I. [1989] "Pharmaceutical pelletilizationtechnology," In J. Swarbrick (ed.) Drugs and the pharmaceuticalsciences: Vol. 37. Pharmaceutical pelletilization technology, New York:Marcel Dekker). Liquid is supplied at low pressure and droplets areformed at the tip of the nozzle by ultrasonic frequency. However, it hasnot been possible to atomize the higher viscosity alginates.

Cellulose acetate phthalate (CAP) is a polyelectrolyte containingionizable carboxyl groups. It is an enteric coating widely used in theindustry for coating tablets. Enteric coatings protect the drug from thegastric juices (pH range 1-6) (Yacobi, A., E. H. Walega [1988] Oralsustained release formulations: Dosing and evaluation, Pergammon Press).CAP serves this purpose by being virtually insoluble below pH 6.0.Aquateric is a commercially available pseudolatex with CAP content of≈70%. Other constituents include Pluronic F-68, Myvacet 9-40,polysorbate 60 and ≦4% free phthalic acid (McGinity [1989], supra). BothCAP and aquateric can be fabricated into microspheresby first dissolvingthem in pH 7.0 distilled deionized water and dropped in acidic solution(Madan, P. L., S. R. Shanbhag [1978] Communications, J. Pharmac. 30:65).Others have used coacervation as the method for microencapsulation(Merkle, H. P., P. Speiser [1973] J. Pharmac. Sci. 62:1444-1448).

The use of various matrices to encapsulate cells and organisms forimplantation in the body has been previously reported (Sun, A. M. [1988]"Microencapsulation of pancreatic islet cells: A bioartificial endocrinepancreas," In Mosbach, K. (ed) Methods in enzymology: Vol 137, AcademicPress, Inc.). Pancreatic cells have been utilized in vitro and in vivofor the production and delivery of insulin. Long term in vivo (in rats)studies of alginate microcapsules containing islet cells, implanted inthe peritoneal cavity, have shown great biocompatibility with no celladhesion to the capsules and a reversal to normal of the previouslydiagnosed diabetic rats (Sun, A. M., Z. Cai, Z. Shi, F. Ma, G.M. O'Shea[1987] Biomaterials, Artificial Cells, and Artificial Organs15(2):483-496).

In vitro cell cultures of hybridomas are now routinely utilized for thepreparation of monoclonal antibodies of great specificity. Cancer celllines are used in vitro for formation of such hybridomas, and also forthe screening and testing of potential carcinogenic and anticarcinogeniccompounds. Also, the industrial utilization of isolated immobilizedcells has received attention, since these can be used as catalysts forbiochemical reactions, and such reactions can be used as important toolsin syntheses and analytical determinations.

In many instances, the direct introduction of a foreign cell into a hostcan produce severe immune response in the host. For example, whengrowing hybridoma cells in the ascites fluid of a host such as a mouse,the mouse has to be pretreated to prevent immune response. Wheninjecting whole islet cells into a human, immune response is also acomplicating factor. A need therefore exists for improved methods offacilitating the introduction of such cells into a host, as well asgenerally for facilitating the manipulation of cells in vitro. Theproblems confronted by the practitioner in attempting to extend many ofthe prior art techniques to the encapsulation of living cells or othersensitive biomaterial are numerous. Many of the existing techniquesoperate under conditions which are too drastic for the survival orcontinuing viability of a living cell, or cause degradation of thebiomaterial desired to be encapsulated. For example, the use of organicsolvents, high temperatures, reactive monomers, cross linkingconditions, and the like, may hamper the viability or otherwise degradethe biomaterial to be encapsulated. Moreover, it is crucial to preventdehydration or osmotic rupture of the cell. Another serious problem isthe necessity of providing the microcapsule walls with sufficientpermeability for nutrients, and secretion and excretion products, topass through, yet prevent the entry of molecules or cells of a host, forexample, products of the hosts immune response, which could destroy theencapsulated material. A further complicating factor is the need toprovide sufficient structural integrity of the capsule while keeping theabove considerations in mind. Prior art methods of alginateencapsulation, while gentle to the encapsulated material, have failed toproduce a capsule of sufficient strength to maintain structuralintegrity over a long period of time.

A need therefore continues to exist for a method to encapsulate livingcells and other sensitive biomaterial under sufficiently mild conditionswhich allow the cells or biomaterial to remain substantially unaffectedby the encapsulation process, yet which also allow the formation of acapsule of sufficient strength to exist over long periods of time.

BRIEF SUMMARY OF THE INVENTION

The subject invention relates to materials and methods for immobilizingbiomaterial such as animal or plant cells, bacteria, algae, fungi,antibiotics or other drugs, viruses, or polypeptides by encapsulation ina polymeric matrix. In accordance with the teachings of the subjectinvention, sensitive biomaterial can be encapsulated with full viabilityand with retained growth ability.

The compositions and methods of the subject invention are illustratedherein with reference to encapsulation of certain bacteria. Bacteriawhich produce a useful product can be encapsulated and introduced into ahuman or other animal. The encapsulation of bacteria is specificallyexemplified herein by the encapsulation of Oxalobacter formigenes. Theencapsulated O. formigenes can be introduced into a human or animal andthe bacteria continue to produce and release enzyme but are not subjectto attack from the immune system.

In specific embodiments of the subject invention, oxalate-decomposingbacteria and enzymes can be successfully encapsulated in either alginatemicrocapsules or cellulose acetate phthalate (CAP) microspheres.Procedures using CAP encapsulation techniques to coat materials such asaspirin and antibiotics for enteric use are also described. Viabilitytests of the encapsulated biomaterials exhibited activity afterencapsulation in either alginate or CAP. For enteric administration,double-encapsulation of CAP in polyvinylpyridine (PVP) can be utilizedto ensure total protection of the enzyme from food substances andthrough the gastric region. This also eliminates the need for forcefeeding animals, for example, during the course of the in vivo studies.One aspect of the invention is the discovery of a procedure foraffecting this double encapsulation. Specifically, by dissolving PVP inmethylene chloride, it is possible to coat CAP microspheres.

Long term studies demonstrated enzyme activity after two months at 37°C. for alginate encapsulated Oxalobacter formigenes. Limitations onbacterial loads were eliminated by reinforcing the shell withalternating layers of pure alginate and poly-L-lysine (PLL). Therefore,therapeutic dosage rates of Orxalobacter formigenes can now beencapsulated.

A further aspect of the subject invention is a procedure for formingsmall alginate microcapsules of about 30-50 μm. The droplets can beformed using, for example, an ultrasonic nozzle and, according to thesubject invention, their entry into a gelling solution can befacilitated by the use of a surfactant and by imparting a surface chargeto these droplets.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of alginate encapsulation using adouble-barrel coextrusion nozzle. Droplets at the end of the nozzle areblown off the needle tip by the coaxial air stream and collected incalcium chloride solution.

FIG. 2 shows piezoelectric sonication of Keltone LV to makemicrocapsules of 30-50 μm in diameter.

FIG. 3 demonstrates size ratio of CAP microspheres to gas flow rate. Wetand dry correspond to the shrinkage before and after drying.

FIG. 4 shows dye (MB) release from CAP microspheres at pH 1.45 and 7.0.

FIG. 5 shows how solvent evaporation technique was used to encapsulateCAP microspheres of 30-50 μm in PVP.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention concerns novel materials and methods formicroencapsulation of biological materials. For example, islet cells orbacteria can be encapsulated according to the procedures of the subjectinvention. The encapsulation structures produced according to thesubject invention have greatly enhanced stability so that thesemicrocapsules can maintain their structural integrity for long periodsof time.

In one embodiment of the invention, the enhanced structural stability ofthe encapsulated cell arises as the result of an improvement overtraditional alginate microencapsulation procedures wherein theimprovement comprises coating the surface of poly-L-lysine (PLL) treatedalginate microcapsules with several layers of pure (bacteria-free)alginate and additionally complexed with more lysine before a finalliquefaction step (citrate treatment, for example).

A standard procedure for producing alginate microcapsules is shown inFIG. 1. Bacteria or other cells are initially placed in an alginatesolution. Droplets from this mixture are then introduced into a solutioncomprising metal ions which stabilize the alginate layer. Metal cationsthat react with and cause gelation (stabilization) of sodium alginatesolutions are, for example, the cations of calcium, barium, zinc,copper, aluminum, and mixtures of these. A water soluble calcium saltsuch as calcium chloride is generally preferred for the process of thisinvention because it is usually non-toxic to living cells. An effectiveconcentration range of calcium dichloride gellant bath, also called thesalt solution or gelling solution, is 1% to 15% (w/v), but 2% to 5% ispreferred. Gelation proceeds further as the concentration of the saltsolution is increased.

There are a variety of methods known in the art for introducing thealginate mixture into the calcium chloride solution. Preferably, themethod used will create droplets of appropriate size which are thenintroduced to an appropriate depth into the gelling solution. Forexample, appropriate droplets can be made using an apparatus which has adouble lumen drop former (Sefton, M. V., R. M. Dawson, R. L. Broughton[1987] Biotechnology and Bioengineering 29:1135-1143). Air is driventhrough the outer lumen and the polymer solution is flow-regulatedthrough a syringe-tip inner lumen.

One aspect of the invention is the use of piezoelectric techniques fordroplet formation combined with electrostatic charging of the dropletsas the droplets fall towards the gelling solution. This technique ispreferably combined with the use of surfactants, as described herein,which help the very small droplets to enter the gelling solution to anappropriate depth. Advantageously,as described in detail herein, a teslacoil can be utilized to impart the necessary charge on the dropletsbefore the droplets enter the gelling solution (see FIG. 2). Afterimmersion in the gelling solution, the microcapsules can be removed fromthe solution and treated with a solution of a positively chargedpolyelectrolyte such as poly-L-lysine (PLL). Typically, a final step inthe process of making alginate microspheres involves removal of thecalcium ions by, for example, treatment with sodium citrate. This stepessentially liquefies the interior of the microcapsule leaving a thinalginate-lysine membrane surrounding the cells which are then in a fluidenvironment.

In addition to the use of electrostatic charging techniques tofacilitate the entry of very small droplets into the gelling solution, afurther aspect of the invention is the production of highly stablemicrocapsules by adding layers of pure alginate and poly-L-lysine beforethe liquefaction step. Thus, in a preferred embodiment of the subjectinvention, alginate microcapsules which have already been treated withPLL are dipped into a pure alginate solution (no bacteria) and rinsedwith saline. The alginate solution at this step is typically diluted toabout 10% of the concentration of the original alginate solution.Isotonic saline can be used to make this dilution. The original alginatesolution is typically about 0.5% to about 2.5% and, preferably, about1.6%. Thus, the concentration of the second alginate solution can beabout 0.05% to about 0.25% and, preferably, about 0.16%. Theseconcentrations can be varied by a person skilled in this art, using theteachings provided herein, to produce microcapsules having the desiredproperties. The concentrations may be varied, for example, to take intoaccount different molecular weights of the alginate solutions.Preferably, the microcapsules are dipped in the alginate solution forabout 2 minutes. Once the microcapsules are removed from the alginatedip, they are then dipped in a poly-L-lysine solution. The concentrationof the poly-L-lysine solutions can be, for example, from about 0.05% toabout 0.3% and, preferably, from about 0.1% to about 0.2%. Preferably,the poly-L-lysine dip is for about 2 minutes. In a preferred embodimentof the invention, the alginate and poly-L-lysine dips are repeated about3 times. In a preferred embodiment of the invention, the alginate dip isperformed last to ensure a biocompatible surface.

There are several variables which can be manipulated to achieve desiredalginate properties (permeability, membrane thickness, and toughness).These variables, which are listed in Table 1, include poly-L-lysine andalginate molecular weight, concentration, and treatment time. The higherthe poly-L-lysine MW, the weaker the microcapsules, since the diffusioninto the alginate is limited. Besides using lower molecular weightpoly-L-lysine, higher concentration and longer treatment time can beused to increase strength.

                                      TABLE 1                                     __________________________________________________________________________    Summary of variables affecting alginate microcapsule properties.              PARAMETERS IN ALGINATE MICROENCAPSULATION                                                               MEASUREMENT PROPERTY                                        Parameter  CHANGE            Shell                                            Manipulated                                                                              Higher/Lower                                                                         Permeability                                                                        Toughness                                                                          thickness                                __________________________________________________________________________    Alginate                                                                              Molecular weight                                                                         Larger MW                                                                            Decreased                                                                           Increased                                                                          Decreased                                Bacteria load                                                                         mg bacteria/ml alginate                                                                  Larger load                                                                          N/A   Decreased                                                                          N/A                                      Poly-L-Iysine                                                                         Molecular weight                                                                         Larger MW                                                                            Increased                                                                           Decreased                                                                          Increased                                Citrate treatment                                                                     Treatment time                                                                           Longer time                                                                          Increased                                                                           Decreased                                                                          No effect                                __________________________________________________________________________

These parameters, and others listed above, can be modified by a personskilled in this art using the teachings provided herein to vary shellthickness, permeability, texture, and toughness of the resultingmicrocapsules.

Kelmar and Keltone are two commonly available alginate preparations.Kelmar (0.8 and 1.6%) and Keltone LV (1.6 and 1.8%) were tested fortheir proper ties with respect to the production of microcapsules. Thecapsules did not show any distinguishable difference other than theirsolution viscosity. Although the higher viscosity Kelmar is expected toyield a tougher shell (Thies, C. [1987] How to make microcapsules: Ionicgelation, Washington University, Biological Transport Laboratory, pp.1-25), we found Keltone LV to be the preferable alternative forprocessing reasons. The 1.6% Kelmar (120-150 kD MW) was very viscous(1390 cps) and extrusion through <22 gauge needle was possible only atvery low flow rates (<2 ml/hour). The 0.8% Kelmar provides fasterextrusion but a weaker capsule with less toleration to the citratetreatment. The Keltone LV (50-80 kD MW) proved to be the betteralternative with easier processibility and good capsular strength. Otherthan the lower molecular weight, the lower calcium content of theKeltone LV (0.2% compared to 1.2% for Kelmar) could be responsible forthe lower viscosity.

There is a weakening effect (more ruptured microcapsules present) as thebacterial load in the capsules is increased. The limit was reached at aload of 20 mg of bacteria/ml of 1.6% Keltone LV solution, at which limitcapsules of spherical shape and moderate strength were impossible tomake. Even at 10 mg/ml, the capsules had a small tail and could not betreated with citrate.

By encapsulation in a permeable polymeric matrix (such as alginate),organisms such as islet cells or Oxalobacter formigenes (anoxalate-degrading bacteria), are isolated from the body's immuneresponse. At the same time, the body is protected from the toxic effectsof the bacteria The matrix is totally impermeable to large moleculessuch as immunoglobulins. Meanwhile, smaller molecules such as glucose oroxalate and their byproducts, freely diffuse in and out of theprotective shell.

In one embodiment of the subject invention, we encapsulate Oxalobacterformigenes in a polymer matrix which is inert in the peritoneal cavity.The matrix must be biocompatible and permeable to oxalate and itsby-products while keeping the bacteria safe from the body's immuneresponse. An ionic gelatin procedure using potassium alginate andpoly-L-lysine was used. The alginates were purchased from Kelco Inc.,and poly-L-lysines (22, 130, 190, 230 kD MW) were purchased from SigmaChemical Company. A bacteria-alginate mixture was made formicroencapsulation. The procedure involved complexing alginate withcalcium ions to form a gel. These microspheres were then treated withpoly-L-lysine to form a more durable outer shell. These microsphereswere transformed into microcapsules by using 1.5% w/v isotonic citratebuffer to liquify the calcium-alginate core.

The capsules of the subject invention are especially useful for theadministration of cells to an animal, wherein the immune response of theanimal towards the cell is to be minimized. Thus, hybridoma cells can begrown in an animal by injecting capsules comprising said cells into theanimal. Drug delivery to an animal can be greatly facilitated byinjecting the animal with capsules comprising cells which produce apharmacologically active agent. Thus, recombinant bacteria which produceinsulin, growth hormone, or interferon, for example, could be injectedinto an animal to proved a ready and continuous source of these activeagents. Encapsulated pancreatic cells could be injected into a diabeticsubject so as to provide a ready source of insulin. Cells which produceantibodies, enzymes, and other bioactive materials can also beadministered. The tough shell and small size of the capsules of thesubject invention facilitate administration of the microcapsules byinjection.

The administration of the capsules could be by local administration,orally, or parenterally.

Alternatively, the capsules of the subject invention have other uses.For example, they can serve as catalytic materials in place of theheretofore used immobilized microbial cells or immobilized enzymes. Theycan be used for analytical purposes, for example, by utilizingdegradative encapsulated microorganisms to release a product such asoxygen, which can then be monitored by means of an oxygen electrode, asis well known in the art When the microcapsules are usedtherapeutically, the amount to be provided to the subject will depend onthe age, sex, size, condition of the subject, other concurrentadministration, contraindications, and the like. For example, it isreadily calculable, for a given application, how much insulin should bereleased into the system over a given period of time, and accordinglythe appropriate amount of encapsulated cells to administer.

In another embodiment of the invention, cells, drugs, or other materialscan be treated with cellulose acetate phthalate (CAP) for enteric use.An important aspect of the subject invention is the discovery of aprocess for coating materials with both CAP and polyvinylpyridine (PVP).This combined coating is highly advantageous for materials which are tobe administered as part of a food substance and, in particular, as partof a food substance with a high moisture content. The double coating isnecessary because CAP, which withstands the highly acidic gastricenvironment, dissolves in neutral pHs such as those of baby food oranimal food pastes. By coating CAP microspheres-with PVP, we havecreated microspheres resistant to both the neutral pHs of foodstuffs aswell as the acidic environment of the stomach.

Following are examples which illustrate procedures, including the bestmode, for practicing the invention. These examples should not beconstrued as limiting. All percentages are by weight and all solventmixture proportions are by volume unless otherwise noted.

EXAMPLE 1 Microencapsulation Procedure

A 1.6 weight percent (wt. %) solution of alginate was prepared bydissolving 1.6 grams of Keltone LV (Kelco Inc., San Diego, Calif.) in100 ml isotonic saline (Celline, Fisher Scientific). The Oxalobacterformigenes bacteria was dispersed in 10 ml of the 1.6 wt. % alginatesolution. The bacteria content (load) was varied between 0.5 mg/ml to 10mg/ml of alginate. The values reported are based on dry weight of thebacteria The bacteria was lyophilized prior to encapsulation. The drybacteria weighs ≈1/4 the wet, unlyophilized bacteria.

A 200 ml solution of 1.4 wt. % CaCl₂ was prepared and buffered with 13mM HEPES to pH 7.2. The alginate/bacteria suspension was extrudedthrough the inner lumen of a double device into the stirred CaCl₂solution. Alginate extrusion rate of 12 ml/hour through a 22 gauge (22G)needle gave the best results. The outer lumen gas flow rate was set at 3liters/minute to form microspheres and provide a drop fall distance of9.0 centimeters. These conditions resulted in spherical (little or notail formation) microspheres with an average diameter of about 700 μmwith very narrow size distribution (675-725 diameter size). The slurry(about 10 ml) was transferred into a filtration unit using a 420 micronTEFLONON® (a fluorocarbon polymer composed of --CF₂ CF₂ --units) mesh asthe filter. This allowed for rapid drainage of solution for timedtreatments of the microcapsules. The bead slurry was washed twice with20 ml of isotonic saline solution. Then the beads were treated with 20ml of 0.1 wt. % poly-L-lysine (22 kD MW, Sigma) in saline. After fiveminutes the solution was drained. The alginate microspheres were thentreated for five minutes with 20 ml of 0.2% CHES in saline. Afterdecanting the CHES the beads were treated with 20 ml of a dilutedalginate solution (about 1/10 the concentration of the extrudedalginate, 0.16% Keltone LV). The beads were washed with isotonic salinesolution before the liquefaction treatment. The beads were suspended in1.5% sodium citrate in 0.45% saline for five minutes. Finally, themicrocapsules were washed and stored in isotonic saline solution at 4°C. for future use.

EXAMPLE 2 Surface Modification of Alginate Microcapsules

The surface of the alginate microcapsules containing bacteria was coatedwith more alternating layers of pure alginate and poly-L-lysine(PLL).This yielded a stronger shell that could withstand the 1.5% citratetreatment for longer periods. The following procedure was performedbefore the citrate treatment:

1. The microencapsulation procedure was performed without the citratetreatment.

2. Prepared a 0.16% Kelmar solution.

3. Prepared 50 ml of 0.2% PLL solution using a combination of differentMW poly-L-lysines (total 100 mg PLL in 50 ml of isotonic salinesolution):

a. 25 mg of PLL, MW=22,400

b. 25 mg of PLL, MW=40,750

c. 25 mg of PLL, MW=100,500

d. 25 mg of PLL, MW=289,000

4. Dipped the microcapsules in the PLL solution for two minutes, removedPLL and washed with saline.

5. Dipped the microcapsules in 0.16% Kelmar for two minutes, washingwith isotonic saline solution after removal.

6. Repeated steps 4 and 5 three times. The alginate dipping was donelast to ensure a biocompatible surface, since exposed PLL sites maycause an inflammatory response.

7. The citrate treatment was performed.

Alginate microcapsules coated with this procedure showed greatertolerance. Microcapsules having a bacteria content of 0.5 mg/mlwithstood citrate treatment longer than 45 minutes with no signs ofweakening or rupture. Without this coating process, capsules rupturedwithin 45 seconds of the citrate treatment.

This process was needed since capsular breakage was observed duringcitrate treatment when bacteria was incorporated. Also, severe limitswere imposed on the bacteria load by the more frequent ruptures as theload concentrations were increased. This procedure offered severalbenefits:

1. Alginate microcapsules coated with this procedure showed greatertolerance to citrate treatment.

2. Larger bacteria loads (>1 mg bacteria/ml alginate solution) can beencapsulated to meet therapeutic dosage rates of oxalate-degradingenzymes required to offset hyperoxaluria

3. Using different combinations of poly-L-lysines, microcapsulepermeability can be customized.

4. The surface is covered with several layers of pure alginate toprevent any inflammatory response due to exposed bacteria sites.

5. The tougher shell may reduce capsular rupture during injection.

This procedure is essential for microcapsule survival in vivo. Thetreated alginate it microcapsules (0.5 mg bacteria/ml 1.6% Keltone LVsolution) withstood citrate treatments longer than 45 minutes withoutdeveloping leaks. However, the untreated capsules of the same batchleaked 45 seconds after adding the citrate.

EXAMPLE 3 Poly-L-Lysine Membrane Thickness of Alginate Microcapsules

Several techniques were attempted to find the thickness of thepoly-L-lysine-alginate membrane. Alginate has a water content of ≈93%(w/w, Goosen, M. F. A., G. M. O'Shea, H. M. Gharapetian [1985]Biotechnology and Bioengineering 27:146-150). Any drying distorted theshape and size of the capsules and resulted in a shrunken mass of solidmaterial. Even lyophilization and critical point drying causeddistortion. Immobilization of the capsules in gelatin was notsuccessful. Finally, freezing the capsules and slicing them with amicrotome showed cross sections of the capsules. The cross sectionsshowed no or minute change in membrane thickness in respect topoly-L-lysine treatment time between 10 and 15 minutes. Our finalarrangement of 0.1% w/v of 22.8 kD poly-L-lysine for 10 minutes showed athickness of 30-40 μm.

EXAMPLE 4 Piezoelectric Sonication of Alginate and ElectrostaticCharging of Droplets

It was desired to make smaller alginate microcapsules. With thepiezoelectric sonicating nozzle (FIG. 2), droplets of 30 μm diameterwere formed using 1.6% w/v Keltone LV. However, these droplets did notenter the calcium chloride solution and instead coated the surface as afilm. Several nonionic surfactants were used to solve this problem. But,surfactant concentrations as high as 10% did not prevent the filmformation.

To keep the droplets separate long enough to penetrate the gellingsolution, some means of surface modification was needed. Electrostaticsurface charging was chosen for this purpose. Using a tesla coil as thepower source, a high voltage electric field was formed to charge thesurface of the droplets. Droplets of alginate were sonicated through anO-ring attached to the tesla coil and into the calcium chloride solution(see FIG. 2). As a person skilled in the art would recognize, a varietyof sources of an electrified field could be utilized to impart thesurface charge to the droplets. We used a "Vacuum Lock Detector" asdescribed at page 1287 of 1991-1992 Fisher Scientific catalog. Thisinstrument was connected to an O-ring as described herein. Using thisapproach, alginate microcapsules of 35 μm average diameter were made.

The following parameters can be used:

1. Nozzle to metal O-ring distance of 3.2 cm (O-ring was attached totesla coil and situated directly under the nozzle).

2. O-ring to CaCl₂ solution distance of 1.8 cm.

3. A 2.0% v/v solution of TWEEN/20 in CaCl₂ (1.37%).

4. 12 ml/hour of alginate pumped through the nozzle.

5. Atomizer power source set to 4.3 watts. 6. 1.6% Keltone LV alginatesolution.

Increasing the alginate pump rate is recommended as a means to achievebetter size distribution of microcapsules. Also, a new filtrationtechnique was developed to ensure proper timing of the treatments. Thecapsules were centrifuged at 750 rpm for five minutes and thesupernatant decanted. By this procedure, it was possible to collect themicrocapsules from solutions in time. A bacterium concentration of 0.5mg/ml (bacteria/alginate solution) was successfully encapsulated andtreated with poly-L-lysine.

In a preferred embodiment, both 2% v/v surfactant and the tesla coil areutilized for this procedure. Using this procedure enabled production ofalginate at a faster rate (>12 ml/hour compared to <2.0 ml/hour withextrusion procedures).

EXAMPLE 5 Cellulose Acetate Phthalate (CAP) Microencapsulation

Microspheres were prepared by extrusion of a CAP mixture (pH 7.5) into5.0 M HCl solution containing one drop of the ionic FC-99, 25%Fluorosurfactant (3M Corp.).

Eight grams of CAP (Polysciences) was suspended in 100 ml of distilleddeionized water (DDI) and dissolved by adjusting the pH to 7.5 usingNaOH. The bacteria load was blended in. This solution was extruded into200 ml of 5 M HCl solution containing 1.0 ml of TWEEN/20(Polyoxyethylene sorbitan monolaurate) and 100 μl of ARLACEL™20(nonionic surfactant, Sorbitan monolaurate, ICI, Inc.). CAP extrusionrate of 12 ml/hour through a 22G needle gave the best results. The gasflow rate was set at 3 liters/minute and a drop fall distance of 9.0centimeters. The resulting microspheres were filtered and washed withDDI water and set under vacuum to dry.

Aquateric (from FMC Inc.) is an alternative CAP containing (70%) aqueousenteric coating by FMC with surfactants and plasticizers incorporated.CAP microspheres decrease in size and weight upon drying (Table 2). Theresults of size distribution upon varying gas flow rates is shown inFIG. 3. The gas flow rate was measured using Manostat gas flow meter.The size of the CAP microspheres was measured before and after drying.The size was determined using an Olympus optical microscope scale.Microspheres obtained at or above 4.54 l/minute had considerabledeformities and a wide size distribution.

In Table 2, dry and wet sizes of two samples at each flow rate arereported. An approximately 1:2 dry:wet size ratio is observed.

                  TABLE 2                                                         ______________________________________                                        Size ratio of CAP microspheres to gas flow rate.                              GAS FLOW                                                                      (liters/minute)                                                                            0.9      1.8      3.2    3.7                                     SAMPLE  SAMPLE   CAP SIZE CAP SIZE                                                                             CAP SIZE                                                                             CAP SIZE                              STATUS  NUMBER   (mm)     (mm)   (mm)   (mm)                                  ______________________________________                                        WET     1        1.50     1.06   0.72   0.64                                          2        1.46     1.04   0.70   0.66                                  DRY     1        0.71     0.44   0.34   0.31                                          2        0.74     0.48   0.31   0.30                                  ______________________________________                                         Shrinkage of CAP microspheres after drying                                    Wet/dry size ratio = 2                                                        CAP extrusion rate = 12 ml/hour                                               9.0 cm drop distance                                                          22 G extrusion needle                                                    

EXAMPLE 6 Piezoelectric Sonication of CAP Microspheres

A piezoelectric sonicating nozzle was used to make smaller capsules. Thebest results were obtained at 2.5 watts (power source) for 0.5 ml/minuteliquid flow or 7.5 watts at 4.0 ml/minute of 8% CAP solution. CAP issoluble at pH greater than 6.0. By maintaining the HCl solution at lessthan pH 2.0, microspheres of 30 to 70 μm in diameter were obtained.

EXAMPLE 7 Tesla Coil

The use of a tesla coil and a combination of surfactants yielded CAPmicrospheres of 30-50 μm. The use of surfactants proved beneficial.Using a combination of TWEEN/20 and ARLACEL™20 (HLB values of 16.7 and8.6, respectively) in the 5.0 M HCl solution and later as a wash inwater during filtration reduced agglomeration very effectively. Usingwater alone (no surfactants) as wash resulted in some aggregation.However, these could easily be broken down to single spheres. The use ofa tesla coil was not necessary since microspheres were made without itsuse. However, the microspheres made using the tesla coil showed lessagglomeration after drying. These microspheres proved essential for thesuccess of the solvent evaporation technique (see Example 9). Due to thelarge interfacial tension, it was not possible to uniformly disperse thelarger (700 μm average diameter) CAP microspheres in the PVP solution.The smaller microspheres alleviated this problem.

The use of a tesla coil allowed for higher liquid flow rate (12 ml/hourat 4.5 watts), without droplets recombining.

EXAMPLE 8 Dye Release From CAP Microspheres

The procedure above was used to make CAP microspheres with 1.25%methylene blue dye incorporated (100 mg MB/8 g CAP). UsingUltraviolet-visible (UV-Vis) spectrometer, the peak maximum for MB wasdetermined at 663 nanometers. Dye release was examined (FIG. 4) at pH1.45 and 7.00 by placing 1.2 mg of CAP microspheres in 1 ml cuvettes.Absorbance was recorded at 663 nanometers for up to 20 minutes.

FIG. 4 shows the dye release from CAP microspheres at pH 1.45 and 7. AtpH 1.45, dye release was minimal. However, at pH 7, the spheres weresolubilized within 10 minutes.

This study demonstrated the ability of CAP microspheres to protect anencapsulated material. The microspheres released minimal amounts of dyewhile at simulated gastric pH (1.45), whereas the dye was releasedrapidly when placed in pH 7 solution.

EXAMPLE 9 Polyvinvypyridine (PVP) Coating of CAP Microspheres

For administering of microencapsulated materials to a human or animal,it can be desirable to incorporate CAP microspheres into food as apaste. Since food paste exposes microspheres to a pH range of 5.0-9.0,it is necessary to protect CAP from dissolving in pH higher than 5.0 bycoating the microspheres with a water impermeable polymer. This polymershould be soluble in acidic pH to ensure solvation in the gastricregion. We have found that polyvinylpyridine (PVP) can be used to coatthe CAP microspheres. The PVP may be poly-2-vinylpyridine. Such doublecoating would be effective when administering a pediatric medicine.Aspirin and antibiotics are examples of such medicines. In a doublecoating of CAP and PVP, the microencapsulated material could be mixedwith baby food and given orally without the enteric coating being tooeasily solubilized.

Three solvent systems were examined for PVP coating of CAP microspheres:

1. HCl (pH 1.0).

2. 15% acetone/methanol mixture.

3. Methylene Chloride.

Casting a film of CAP microspheres in a pH 1.0 HCl solution of PVPresulted in a sheet of coated CAP microspheres which could be brokendown for administration. This procedure was not desirable since anunknown amount of CAP microspheres would be exposed due to powderizationof the casted PVP sheet. Also, the slow rate of evaporation of HClsolution and the small yield of coated CAP hindered further pursuit ofthis procedure.

PVP was also dissolved in a mixture of 85% methanol and 15% acetone.However, this solvent system dissolved the microspheres as well.Regardless, a film was cast and the solvent evaporated quickly enough toleave some CAP microspheres intact This film was introduced to acidicmedia (pH 1.0) and the PVP film dissolved within five minutes, leavingthe CAP microspheres free-flowing. The pH was then raised to 7.5. PVPreprecipitated at once but did not interfere with CAP dissolving (within10 minutes). Since an outline of reprecipitated PVP in the shape of CAPmicrospheres was left, the pH was reduced to 1.0, which left a clearsolution. This removed doubts of residual CAP remaining unsolved.Therefore, the PVP protected the CAP microspheres at higher pH and didnot interfere with solvation of the microspheres after reprecipitation.

Attempts to find a preferred solvent yielded methylene chloride.

Solvent Evaporation:

1. Make 250 ml of 0.25% (w/v) solution of Airvol 205 (PVA, Air Products)in water.

2. Dissolve 0.8 g 200 kD PVP (poly-2-vinylpyridine, Aldrich) in 10 mlmethylene chloride.

3. Add about 80 mg of CAP microspheres (30-50 μm diameter) and stir forfive minutes.

4. While stirring the PVA solution at 400 rpm in a 400 ml beaker, theCAP/PVP suspension is added in a continuous narrow stream.

5. The emulsion is stirred for three hours.

6. Make a 1.0% (v/v) solution of TWEEN/20 in water adding one drop ofARLACEL™20 for every 1.0 ml of TWEEN/20 used.

7. After three hours, dilute the emulsion with the TWEEN/20 solution andvacuum filter the spheres washing excessively to remove the PVA.

8. Vacuum dry the cake.

By using the solvent evaporation technique, it is possible to suspendthe CAP microspheres in the PVP solution (dissolved in methylenechloride) and emulsify this solution complex in water. After evaporationof the solvent (methylene chloride), the PVP coated CAP microspheres canbe harvested for oral administration (see FIG. 4).

For this procedure an emulsifier was needed. PVA (polyvinyl alcohol) waschosen as the emulsifier. Elvanol 50-42 (du Pont) and Airvol 205 (AirProducts) were used. The 0.25% Elvanol 50-42 solution resulted in majorclumping. After several attempts changing different variables, the useof this emulsifier was stopped. After switching to Airvol 205, thequality of the emulsified phase was better, giving more uniform dropformation and much less clumping. The clumping was also reduced by usinghigher stirring rate (400 compared to 300 rpm) to keep the PVA wet.Methylene chloride has a solubility of 2 g/100 g water. This can be usedto remove any residual solvent when harvesting by washing with excessvolume of water. The optimum harvesting was done after three hours ofstirring. Any less time could result in recombination of the PVP spheresdue to the presence of solvent. Any longer period is unnecessary andcould result in more clumping. It is believed that the clumping (mostlyon the impeller) could be alleviated by using a TEFLON™ (a fluorocarbonpolymer composed of--CF₂ CF₂ --units) impeller. Harvesting themicrospheres was difficult due to the adhesion of the PVP spheres toglass. Washing the microspheres with a 1% solution of TWEEN/20 andARLACEL™ prevented this problem. After vacuum drying the spheres, thefew clumps present were easily broken to individual microspheres.

Some variables affecting this technique are as follows:

1. Airvol 205 proved to be the emulsifier with the best results (leastamount of debris and aggregation).

2. By using higher rpms, clumping on the impeller can be reduced.

3. If the droplets are harvested before the solvent is completelyevaporated, the droplets may recombine.

4. If PVA is removed too early, agglomeration will occur. If PVA isremoved too late, the load (in this case, CAP) can be released into theaqueous (continuous) phase.

As described above, oral delivery of pH-sensitive proteins requiresprotection from gastric juices. Stomach upsetting agents (analgesics,etc.) have been enteric coated with polymers such as cellulose acetatephthalate (CAP). However, these require extensive formulationdevelopment into tablets and syrups which are undesirable and hard toadminister to infants. With the double encapsulation technique of thesubject invention, tasteless, water-impermeable microspheres can beincorporated into baby food. The outer poly-2-vinylpyridine layer isstable at higher pH, but will dissolve in gastric juices, releasing theCAP microspheres containing the drug. In turn, CAP microspheres willrelease their contents in the intestine.

EXAMPLE 10 Dye Release from PVP-Coated CAP Microspheres

Dye release from PVP-coated CAP microspheres in several solutions ofdifferent pH was tested. The microspheres were put through the followingstages:

1. Microspheres were placed in a pH 8 solution. Solution was removedafter 15 minutes.

2. A pH 2 HCl solution was added and removed after 20 minutes.

3. A solution of pH 1.1 was added for 15 minutes.

4. In the final stage the microspheres were put in pH 8 solution.

No change in the appearance of the PVP microspheres was observed afterintroducing them to the pH 8 solution. In pH 2, PVP microspheres showeda very slow release of CAP microspheres. After 20 minutes, the majorityof the spheres were intact When pH 1.1 solution was added, much fasterrate of dissolution was observed. All the PVP microspheres dissolvedafter 15 minutes, releasing the CAP microspheres which exhibited no dyerelease at this stage. CAP microspheres were totally dissolved withinfive minutes after placing the microspheres in pH 8 solution, releasingthe dye, which in turn colored the reprecipitated PVP crystals.

The microspheres were placed in a simulated process of emulating thefood paste(pH as high as 8), the gastric region (pH 1-2, fasted),intestines(pH 6.5-8.0). The PVP protected the CAP spheres fromdissolving in a pH 8 solution. Afterwards, slow release of CAPmicrospheres was observed at pH 2. A faster CAP release was exhibited atpH 1.1 with all PVP spheres dissolving after 15 minutes. At this pH theCAP microspheres were intact with the dye incorporated, which suggeststhey would have protected the active ingredient after being released.When pH was changed to 8, the CAP microspheres dissolved, releasing thedye (MB), which in turn colored the reprecipitated PVP crystals.Therefore, the system functioned properly.

EXAMPLE 11 Enzyme Activity Studies

Studies were conducted using fresh Oxalobacter formigenes encapsulatedin alginate as described in Example 2. The activity of the freshbacteria was determined to be about 71.1 nmole/min. mg(average of tworuns: 70.8 and 71.4 nmole/min. mg, respectively). This result wasrepeated using varying amounts of bacteria.

We incubated samples of encapsulated bacteria at 37° C. for long-termstudy. Some samples showed signs of contamination (no preservative wasused); however, uncontaminated microcapsules showed a 40% activity aftertwo months and 10% activity after four. These activities are in a rangewhere they can degrade a normal oxalate load in vivo (≈40 mg/day,Hodgkinson [1977], supra).

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

We claim:
 1. In a method for preparing microcapsules of a desiredmaterial wherein said method comprises the following steps:(a) mixingsaid desired material to be encapsulated with a matrix-formingcomposition; (b) making droplets of the mixture of part (a) andintroducing said droplets into a gelling solution to form droplets; (c)treating the gel droplets formed in part (b) with a polyelectrolyte toform a microcapsule;an improvement which comprises the addition of atleast one more polyelectrolyte layer and at least one morematrix-forming layer to the microcapsule of part (c), wherein saidpolyelectrolyte is positively charged and said matrix-formingcomposition comprises alginate, wherein a matrix-forming layer is thelast layer to be added to said microcapsule.
 2. The method, according toclaim 1, wherein part (a) comprises mixing said material to beencapsulated with a solution of alginate having a concentration of about0.5% to about 2.5% of alginate.
 3. The method, according to claim 2,wherein said alginate has a molecular weight of about 50 kD to about 150kD.
 4. The method, according to claim 1, wherein at least one additionallayer of alginate is added to said microcapsule by dipping saidmicrocapsule into an alginate solution wherein said alginate solutionhas a concentration of approximately 10% of the concentration of thealginate solution used in part (a).
 5. The method, according to claim 4,wherein said polyelectrolyte is poly-L-lysine.
 6. The method, accordingto claim 5, which comprises adding three additional layers of alginateand three additional layers of poly-L-lysine with an alginate layerbeing the last to be added.
 7. The method, according to claim 6, whichfurther comprises a liquefaction step.
 8. The method, according to claim7, wherein said liquefaction step comprises treatment with citrate. 9.The method, according to claim 8, wherein said citrate treatment is witha 1.5% citrate solution for at least 5 minutes.
 10. The method,according to claim 9, wherein said encapsulated material is selectedfrom the group consisting of bacteria polypeptides and islet cells. 11.The method, according to claim 10, wherein said bacteria are Oxalobacterformigenes.
 12. The method, according to claim 1, wherein said gellingsolution is a calcium chloride solution.
 13. A microcapsule produced bythe process of claim 1.